Understanding pancreatic secretion is important both as it relates to the function of this digestive organ and in understanding basic cellular mechanisms. Seratogues such as cholecystokinin (CCK) and acetylcholine (ACh) act on the exocrine pancreas to increase cytoplasmic Ca2+ and release digestive enzymes. There is good evidence that the increase in Ca2+ is both necessary and sufficient to drive enzyme secretion. However, there is still imperfect understanding of how free intracellular Ca2+ is controlled and how Ca2+ acts. In this proposal Ca2+ will be measured by dual wavelength fluorescence digital imaging. The mechanism of Ca2+ oscillations in time and Ca2+ waves around an acinus in space will be investigated. The role of Ca2+, IP3 and gap junctions will be evaluated using both focal extracellular stimulation and microinjection. We have also established the existence of [Ca2=+]i oscillations which doe not involve IP3. We will also investigate the importance of two Ca2+ activated enzymes known to be present in pancreas in mediating the action of Ca2+. These enzymes are protein phosphatase 2B (PP2-B) and calcium calmodulin kinase II. In both cases molecular biological techniques (PCR phosphatase 2B (PP2-B) and calcium calmodulin kinase II. In both cases molecular biological techniques (PCR and library screening) will be used to establish the molecular form present in acini. The properties of native and recombinant enzymes will be characterized and antibodies to the proteins generated. Substrate proteins dephosphorylated or phosphorylated by these enzymes will be identified. In both cases we will establish protocols to inhibit the enzyme to establish its importance in regulation protein phosphorylation and secretion. The immune suppressants cyclosporin A and FK506 will be used to inhibit PP2- B. The kinase inhibitor KN-62, autoinhibitory domain peptides and antibody will be evaluated as inhibitors of CaM Kinase II. In addition to biochemical measurements of secretion in intact and permeabilized cells, we will utilize capacitance measurements on single patch clamped acinar cells to study the temporal pattern of secretion and its relation to Ca2+ oscillations. These studies using patch clamped cells will also allow introduction of peptides, proteins, and Ab to ascertain the importance of PP2-B and CaM Kinase II in secretion. These studies are also relevant to understanding of the abnormal secretion seen in pancreatic diseases such as pancreatitis.